表达乳铁蛋白肽的猪源罗伊氏乳酸杆菌对断乳仔猪抗霍乱沙门菌感染的效果分析

doi:10.11843/j.issn.0366-6964.2021.010.018

摘要/Abstract

摘要： 旨在分析表达牛乳铁蛋白肽的重组猪源罗伊氏乳酸杆菌pPG-LFCA-E/LR-CO21作为饲用微生态制剂对仔猪促生长与抵抗猪霍乱沙门菌感染的作用。将该重组菌连续饲喂28日龄断乳仔猪21 d，并设立空菌组、对照组以及抗生素组。第21天时对断乳仔猪连续口服感染猪霍乱沙门菌3 d，并设置7 d的观察期；仔猪感染后采集试验组与对照组血清、肠黏液及肠组织。检测结果显示重组菌组仔猪平均日增重与免疫器官指数均显著提高（P<0.05），料重比极显著降低（P<0.01）。感染猪霍乱沙门菌后，相对于对照组，重组菌组仔猪日增重提高，腹泻率降低；重组菌组仔猪血液中IgG以及肠黏液中IL-4、sIgA的量极显著提高（P<0.01），IL-2、IL-12、IL-6的量显著降低（P<0.05）；重组菌组仔猪肠道紧密连接蛋白ZO-1、Claudin-1的基因转录水平和TLR4、Myd88和MLCK的基因转录水平极显著提高（P<0.01），仔猪肠道内猪霍乱沙门菌的数量极显著降低（P<0.01）；重组菌组与抗生素组无明显差异（P>0.05）。以上结果表明，表达牛乳铁蛋白肽的重组猪源罗伊氏乳酸杆菌pPG-LFCA-E/LR-CO21可以提高断乳仔猪的生长性能、促进肠道形态发育、增强肠道屏障功能，并且在一定程度上可以保护仔猪免受猪霍乱沙门菌的感染，表明重组菌作为微生态制剂替代断乳仔猪的饲用抗生素具有一定的应用前景。

关键词: 乳铁蛋白肽, 重组罗伊氏乳酸杆菌, 猪霍乱沙门菌, 断乳仔猪

Abstract: The purpose of this study was to analyze the effect of recombinant Lactobacillus reuteri from piglets expressing bovine lactoferrin peptide (pPG-LFCA-E/LR-CO21) as microecological on the growth promotion of piglets and resistance to Salmonella Choleraesuis CVCC79102 infection. In this experiment, the recombinant bacteria were continuously fed to 28-day-old weaned piglets for 21 days, and empty bacteria group, control group and antibiotic group were set up. On the 21st day, weaned piglets were orally infected with Salmonella Choleraesuis for 3 days, and the observation period was set for 7 days, and the serum, intestinal mucus and intestinal tissue of the experimental groups and the control group were collected after infection. The results showed that the average daily gain, feed gain ratio and immune organ index of piglets in the recombinant bacteria group were significantly increased (P<0.05). After being infected with Salmonella Choleraesuis CVCC79102, in the recombinant bacteria group, application of the recombinant bacteria increased the daily gain of piglets and reduced the rate of diarrhea compared with the control group; it significantly increased the amount of IgG in blood and IL-4 and sIgA in intestinal mucus (P<0.01). and significantly reduced the amount of IL-2, IL-12, IL-6 (P<0.05). It significantly increased the gene transcription level of intestinal tight junction protein ZO-1, Claudin-1 and the gene transcription levels of TLR4, Myd88 and MLCK, and significantly decreased the number of Salmonella Choleraesuis in the intestinal tract of piglets (P<0.01). There was no significant difference between the recombinant bacteria group and the antibiotic group. The above results show that pPG-LFCA-E/LR-CO21 can regulate the growth performance, intestinal morphology and intestinal barrier function of weaned piglets, and can protect piglets from Salmonella Choleraesuis infection to a certain extent, indicating that the recombinant bacteria have a certain application prospect feeding as microecological for weaned piglets.

Key words: bovine lactoferrin peptide, recombinant Lactobacillus reuteri, Salmonella Choleraesuis, weaned piglets

中图分类号:

S859.797

王雪莹, 高亢, 蔡吉垚, 张森豪, 解伟纯, 王晓娜, 崔文, 姜艳平, 周晗, 王丽, 乔薪瑗, 徐义刚, 李一经, 唐丽杰. 表达乳铁蛋白肽的猪源罗伊氏乳酸杆菌对断乳仔猪抗霍乱沙门菌感染的效果分析[J]. 畜牧兽医学报, 2021, 52(10): 2874-2886.

WANG Xueying, GAO Kang, CAI Jiyao, ZHANG Senhao, XIE Weichun, WANG Xiaona, CUI Wen, JIANG Yanping, ZHOU Han, WANG Li, QIAO Xinyuan, XU Yigang, LI Yijing, TANG Lijie. Analysis of Effects of Lactobacillus reuteri from Piglets Secreting Lactoferrin Peptides against Salmonella Choleraesuis Infection of Weaned Piglets[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2874-2886.

参考文献 40

[1]	岂晓鑫. 1950-2009年动物源沙门菌的分型、耐药性及致病性研究[D]. 扬州:扬州大学, 2018.QI X X. Typing, drug resistance and pathogenicity of Salmonella isolates from animal during 1950-2009 in China[D]. Yangzhou:Yangzhou University, 2018. (in Chinese)
[2]	王克. 猪沙门氏菌病的临床症状与防治[J]. 养殖与饲料, 2020(4):71-72.WANG K. Clinical symptoms and prevention of swine salmonellosis animals[J]. Animals Breeding and Feed, 2020(4):71-72. (in Chinese)
[3]	OSCAR T. Salmonella prevalence alone is not a good indicator of poultry food safety[J]. Risk Anal, 2021, 41(1):110-130.
[4]	张锦, 李庆贺, 郑麦青, 等. 沙门菌感染雏鸡调节性T细胞变化的分析[J]. 畜牧兽医学报, 2019, 50(11):2302-2308.ZHANG J, LI Q H, ZHENG M Q, et al. Analysis of regulatory T cells changes in chicks infected with Salmonella[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(11):2302-2308. (in Chinese)
[5]	LONGO A, LOSASSO C, VITULANO F, et al. Insight into an outbreak of Salmonella choleraesuis var. Kunzendorf in wild boars[J]. Vet Microbiol, 2019, 238:108423.
[6]	MOLINO M G, PÉREZ D R, BLANCO P G, et al. Outbreaks of antimicrobial resistant Salmonella Choleraesuis in wild boars piglets from central-western Spain[J]. Transbound Emerg Dis, 2019, 66(1):225-233.
[7]	宋德平. 新现和再现猪主要肠道腹泻病毒及腹泻猪肠道微生物群落宏基因组学研究[D]. 南昌:江西农业大学, 2016.SONG D P. Studies on Important emerging and re-emerging porcine diarrheal viruses and metagenomics of gut microflora of diarrheal pigs[D]. Nanchang:Jiangxi Agricultural University, 2016. (in Chinese)
[8]	GIRARD M, BEE G. Invited review:tannins as a potential alternative to antibiotics to prevent coliform diarrhea in weaned pigs[J]. Animal, 2020, 14(1):95-107.
[9]	BAQUERO F, NEGRI M C. Strategies to minimize the development of antibiotic resistance[J]. J Chemother, 1997, 9(S3):29-37.
[10]	MENG L X, XU G L, LI J S, et al. Bovine lactoferricin P13 triggers ROS-mediated caspase-dependent apoptosis in SMMC7721 cells[J]. Oncol Lett, 2017, 13(1):511-517.
[11]	TU M L, XU S Q, XU Z, et al. Identification of dual-function bovine lactoferrin peptides released using simulated gastrointestinal digestion[J]. Food Biosci, 2021, 39:100806.
[12]	BIASIBETTI E, RAPACIOLI S, BRUNI N, et al. Lactoferrin-derived peptides antimicrobial activity:an in vitro experiment[J]. Nat Prod Res, 2020:1-5.
[13]	ELNAGDY S, ALKHAZINDAR M. The potential of antimicrobial peptides as an antiviral therapy against COVID-19[J]. ACS Pharmacol Transl Sci, 2020:3(4):780-782.
[14]	WANG Y, BEKHIT A E D A, MASON S L, et al. Lactoferrin isolation and hydrolysis from red deer (Cervus elaphus) milk and the antibacterial activity of deer lactoferrin and its hydrolysates[J]. Foods, 2020:9(11):1711.
[15]	WEINBERG E D. Antibiotic properties and applications of lactoferrin[J]. Curr Pharm Des, 2007:13(8):801-811.
[16]	MILLER F P, VANDOME A F, MCBREWSTER J. Lactobacillus reuteri[M]//Lactobacillus Reuteri. Alphascript Publishing, 2010.
[17]	HAN S K, KIM J K, JOO M K, et al. Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 alleviate Escherichia coli-induced depression and gut dysbiosis in mice[J]. J Microbiol Biotechnol, 2020, 30(8):1222-1226.
[18]	KIM H B, ISAACSON R E. The pig gut microbial diversity:understanding the pig gut microbial ecology through the next generation high throughput sequencing[J]. Vet Microbiol, 2015, 177(3-4):242-251.
[19]	GAO K, WANG C, LIU L, et al. Immunomodulation and signaling mechanism of Lactobacillus rhamnosus GG and its components on porcine intestinal epithelial cells stimulated by lipopolysaccharide[J]. J Microbiol Immunol Infect, 2017, 50(5):700-713.
[20]	段海涛. 高效调质低温制粒畜禽饲料加工工艺及其对生长育肥猪生长性能的影响研究[D]. 北京:中国农业科学院, 2018.DUAN H T. Study on the high-efficiency condition and low-temperature pelleted processing technology and effects on growth performance of growing and fattening pigs[D]. Beijing:Chinese Academy of Agricultural Sciences, 2018. (in Chinese)
[21]	MCORIST S, KHAMPEE K, GUO A. Modern pig farming in the People's Republic of China:growth and veterinary challenges[J]. Rev Sci Tech, 2011, 30(3):961-968.
[22]	YANG Q L, HUANG X Y, WANG P F, et al. Longitudinal development of the gut microbiota in healthy and diarrheic piglets induced by age-related dietary changes[J]. MicrobiologyOpen, 2019, 8(12):e923.
[23]	JAYARAMAN B, NYACHOTI C M. Husbandry practices and gut health outcomes in weaned piglets:a review[J]. Anim Nutr, 2017, 3(3):205-211.
[24]	VANGROENWEGHE F, POULSEN K, THAS O. Supplementation of a β-mannanase enzyme reduces post-weaning diarrhea and antibiotic use in piglets on an alternative diet with additional soybean meal[J]. Porc Health Manag, 2021, 7(1):8.
[25]	印遇龙, 杨哲. 天然植物替代饲用促生长抗生素的研究与展望[J]. 饲料工业, 2020, 41(24):1-7.YIN Y L, YANG Z. Research and prospect of natural plant substitute for antibiotic growth promoters in feed[J]. Feed Industry, 2020, 41(24):1-7. (in Chinese)
[26]	CHEN H S, VELAYUDHAN D E, LI A, et al. Growth performance, gastrointestinal microbial activity, and immunological response of piglets receiving microencapsulated Enterococcus faecalis CG1. 0007 and enzyme complex after an oral challenge with Escherichia coli (K88)[J]. Canadian J Anim Sci, 2016, 96(4):609-618.
[27]	CHAHARDOOLI M, NIAZI A, ARAM F, et al. Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification[J]. J Sci Food Agric, 2016, 96(2):569-575.
[28]	陈雷, 原现军, 郭刚, 等. 添加乳酸菌制剂和丙酸对全株玉米全混合日粮青贮发酵品质和有氧稳定性的影响[J]. 畜牧兽医学报, 2015, 46(1):104-110.CHEN L, YUAN X J, GUO G, et al. The effects of lactic acid bacteria and propionic acid on the fermentation quality and aerobic stability of total mixed ration silages prepared with whole-crop corn in Tibet[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(1):104-110. (in Chinese)
[29]	CANO-GARRIDO O, SERAS-FRANZOSO J, GARCIA-FRUITÓS E, et al. Lactic acid bacteria:reviewing the potential of a promising delivery live vector for biomedical purposes[J]. Microb Cell Fact, 2015, 14(1):137.
[30]	KAUR T, BALGIR P P, KAUR B. Correction to:construction of a shuttle expression vector for lactic acid bacteria[J]. J Genet Eng Biotechnol, 2020, 18(1):38.
[31]	KAUR T, BALGIR P P, KAUR B. Construction of a shuttle expression vector for lactic acid bacteria[J]. J Genet Eng Biotechnol, 2019, 17(1):10.
[32]	王书博, 徐义刚, 陈秋艳, 等. 表达猪圆环病毒2型cap蛋白的重组罗伊氏乳酸杆菌在小鼠诱导的免疫应答分析[J]. 畜牧兽医学报, 2020, 51(9):2238-2249.WANG S B, XU Y G, CHEN Q Y, et al. Analysis of immune response induced by recombinant Lactobacillus reuteri expressing cap protein of porcine circovirus type 2 in mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9):2238-2249. (in Chinese)
[33]	赵东方. 罗伊氏乳酸杆菌的分离鉴定及其抵抗仔猪感染F4+ETEC效果的分析[D]. 哈尔滨:东北农业大学, 2019.ZHAO D F. Isolation and identification of Lactobacillus reuteri and its effection on F4+ETEC infection piglets[D]. Harbin:Northeast Agricultural University, 2019. (in Chinese)
[34]	于淑媛, 冀禹彤, 陈秋艳, 等. 乳酸乳球菌表达重组牛乳铁蛋白肽的抑菌活性分析[J]. 微生物学报, 2021, 61(2):428-443.YU S Y, JI Y T, CHEN Q Y, et al. Antibacterial activity of the recombinant bovine lactoferrin peptide expressed by Lactococcus lactis[J]. Acta Microbiologica Sinica, 2021, 61(2):428-443. (in Chinese)
[35]	YI H B, HU W Y, CHEN S, et al. Cathelicidin-WA improves intestinal epithelial barrier function and enhances host defense against enterohemorrhagic Escherichia coli O157:H7 Infection[J]. J Immunol, 2017, 198(4):1696-1705.
[36]	Gunter Sandra Argyll Biotechnologies LLC. Composition and method for immunomodulation in mammals:US, 6719984B1[P] 2004-04-13.
[37]	KIM J S, ELLMAN M B, YAN D Y, et al. Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc[J]. J Cell Physiol, 2013, 228(9):1884-1896.
[38]	高彦华. 猪抗菌肽PR-39的表达特性和调控及免疫调节机制研究[D]. 杭州:浙江大学, 2014.GAO Y H. The expression characteristics, regulation and the immunomodulatory mechanism of porcine antimicrobial peptide PR-39[D]. Hangzhou:Zhejiang University, 2014. (in Chinese)
[39]	赵雪芹. 抗菌肽类似物JH-3抵御沙门氏菌感染的作用机制研究[D]. 新乡:河南科技学院, 2018.ZHAO X Q. Mechanism of antimicrobial peptide analogue JH-3 against Salmonella[D]. Xinxiang:Henan Institute of Science and Technology, 2018. (in Chinese)
[40]	TANG X S, TANG Z R, WANG S P, et al. Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris[J]. Appl Biochem Biotechnol, 2010, 73(2):132-139.